The object of the proposed research is to identify and characterize the normal low density lipoprotein (LDL) receptors on the plasma membranes of fibroblasts from normal subjects and the defective LDL receptors from plasma membranes of patients with familial hypercholesterolemia (FH). To accomplish this we will use new, very sensitive techniques which we have recently developed to screen membrane proteins for small alterations in primary structure. Our preliminary results suggest that these techniques are capable of detecting differences as small as the modification of a single tryptic peptide residue in two otherwise identical proteins. Only microgram quantities of each protein, isolated as a discrete polyacrylamide gel band, are required. Critical to this approach is our new method for double label thin layer autoradiography. Following identification of the normal and defective LDL binding proteins, each will be characterized structurally and, to some extent, functionally. Structural analysis will include peptide mapping, amino acid composition, amino acid sequencing in the regions of dissimilarity between normal and defective proteins, and analysis for possible post-translational modifications such as phosphorylation or glycosylation. Functional analysis will focus primarily on development of antibodies specific for normal and defective LDL binding proteins. The antibodies will then be used to localize the binding protein within the membrane, to follow its fate after exposure to LDL, and to study its biosynthesis and insertion into the membrane in normal and FH cells. The immediate purpose of this work is to help elucidate the nature of the control mechanisms for regulation of cholesterol biosynthesis with particular emphasis on events occurring at the plasma membrane. Detailed structural comparison of normal and FH binding proteins should answer the question of whether altered LDL binding function in FH is the result of a direct modification of the gene coding for this protein or an indirect effect due to post-translational modification. An additional immediate goal will be to provide antibodies for clinical investigation and early detection of familial hypercholesterolemia. In a more general sense, however, the purpose of this research is to utilize pathology as a probe of membrane protein structure-relationships and to demonstrate the general applicability of our new techniques to similar problems involv (Text Truncated - Exceeds Capacity)